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L C Harrison

Publications and source records attributed to L C Harrison.

At least 127 records · Page 7Linked to original sources
PubMed1992

An ELISA for antibodies to recombinant glutamic acid decarboxylase in IDDM.

To detect serum antibodies to GAD in subjects with IDDM, three recombinant mBGAD 67 peptides encompassing the full-length protein were used in an ELISA. In this study 7 of 9 (78%) preclinical IDDM subjects (ICA+ first-degree relatives of a person with IDDM) and 6 of 13 (46%) recent-onset IDDM subjects, but no subjects with Graves' disease (n = 10) or scleroderma (n = 10), nor healthy nondiabetic control subjects (n = 10) had antibodies that reacted with one or more of the recombinant mBGAD peptides. We found no preferential reactivity with any recombinant peptide. Although only 3 preclinical subjects and 1 recent-onset subject had antibodies to all three mBGAD peptides, the results indicate that mBGAD 67 contains at least three B-cell autoepitopes. Compared with an immunoprecipitation assay of native human brain GAD, the ELISA detected 5 of 6 (83%) preclinical and 6 of 6 (100%) recent-onset IDDM subjects. The ELISA should facilitate screening to evaluate the role of autoimmunity to GAD in the development of IDDM.

Adolescent↗
PubMed1992

Comparison of two commonly used standard IVGTTs.

OBJECTIVE: To compare the magnitude and reproducibility of the FPIR measured during two different IVGTT protocols in nondiabetic subjects. RESEARCH DESIGN AND METHODS: Nine control subjects each had two pairs of IVGTTs with either a 4-min infusion of 0.5 g/kg glucose or a 1-min infusion of 0.3 g/kg glucose. Blood glucose and serum insulin were measured before and 1, 2, 3, 5, and 10 min after completion of the glucose infusion. The FPIR was measured with either 1 + 3-, 2 + 3 + 5-, or 1 + 3 + 5-min serum insulins, areas under the insulin curve (0-5 or 0-10 min), or the ratio of serum insulin to blood glucose area. RESULTS: The FPIR was higher in eight of nine subjects with the short-infusion test, but the within-subject variation of the two methods was identical. Reproducibility was not significantly improved with an integrated insulin area or insulin-to-glucose ratio measurement. CONCLUSIONS: Reproducibility of the FPIR measured during IVGTT is not significantly affected by the duration of the glucose infusion. However, the magnitude of the difference in FPIR observed between the two protocols highlights the need for standardization of the methodology if the IVGTT is to be used in studies of the preclinical stage of IDDM.

Adult↗
PubMed1991

Cloning and partial nucleotide sequence of human glutamic acid decarboxylase cDNA from brain and pancreatic islets.

We report partial nucleotide sequences of the human enzyme glutamic acid decarboxylase (GAD) from brain and pancreatic islets which encode the middle 180 amino acids of GAD. The brain and islet GAD sequences display a high degree of sequence homology with the equivalent region of other mammalian brain GAD cDNAs. Alignment of the brain and islet GAD sequences showed that there were 45 nucleotide differences which, at the translational level, would result in seven amino acid substitutions. These results which suggest that different isomeric forms of human GAD exist in brain and pancreas may be relevant to the pathogenesis of stiff man syndrome (SMS) and insulin-dependent diabetes mellitus (IDDM), respectively, two distinct but associated clinical disorders in which GAD is the target of autoantibodies.

Amino Acid Sequence↗
PubMed1991

Changes in insulin-receptor structure associated with trypsin-induced activation of the receptor tyrosine kinase.

The tyrosine kinase of the insulin receptor can be activated by trypsin treatment. The concomitant abolition of insulin binding has been postulated to result from proteolytic destruction of the receptor. A discrepancy between the decrease in insulin binding and receptor immunoreactivity after trypsin treatment led us to investigate more closely the structure of the trypsin-treated receptor. After trypsin treatment of the CHOT cell line, which over-expresses transfected human insulin receptors, insulin binding was significantly decreased, but reactivity with five alpha-subunit monoclonal antibodies was either unaffected or only moderately decreased, indicating that the alpha-subunit was substantially intact. Examination of receptor structure after trypsin treatment, receptor autophosphorylation and gel electrophoresis revealed a single band at 110 kDa in non-reduced gels, comprising a small fragment (21 kDa) of the alpha-subunit linked to the beta-subunit by class II disulphides. When the receptor was radio-labelled with 125I, two additional alpha-subunit bands of 142 kDa and 81 kDa (composed of identical reduced bands) were observed on non-reduced gels, which contained disulphide-linked (class I) fragments. All fragments could be precipitated by antibodies to both alpha- and beta-subunits. However, only antibodies directed towards the N-terminus of the receptor could immunoblot trypsin-treated fragments. Thus activation of the receptor tyrosine kinase by trypsin occurs after cleavage, but not loss of the alpha-subunit. This finding has implications for the mechanism of transmembrane activation of the receptor kinase by insulin.

Animals↗
PubMed1991

Stimulation of glucose transporter (GLUT1) mRNA and protein expression by inhibitors of glycosylation.

Glucose deprivation increases the steady-state levels of mRNA for the rat brain/HepG2-type glucose transporter (GLUT1) in L6 myocytes. Glucose deprivation also inhibits N-linked glycosylation. We therefore investigated a possible relationship between inhibition of glycosylation and GLUT1 expression in cultured L6 myocytes by determining the effects on GLUT1 expression of known inhibitors of glycosylation, namely tunicamycin, 2-deoxyglucose and glucosamine. All conditions prevented incorporation of [3H]mannose into TCA-precipitable myocyte protein and resulted in a 2- to 5-fold increase in the level of GLUT1 mRNA detected on Northern blots. Glucose deprivation and tunicamycin treatment caused an approx. 2-fold increase in GLUT1 mRNA half-life. GLUT1 protein, detected on immunoblots, accumulated 10- to 20-fold in response to all glycosylation inhibitors, with apparent molecular masses of 40 kDa after glucose deprivation, 42 kDa after 2-deoxyglucose and 38 kDa after glucosamine or tunicamycin treatments, compared to 45-50 kDa in glucose-fed cells. However, glucose deprivation was the only condition in which the rate of 2-deoxy-[3H]glucose uptake increased (3- to 5-fold). These results demonstrate a direct correlation between inhibition of glycosylation and the induction of GLUT1 mRNA and protein expression and suggest that the stability of GLUT1 mRNA is controlled by a signal associated with glycosylation.

Animals↗
PubMed1991

Detection of cytomegalovirus by the polymerase chain reaction. A simple, rapid and sensitive non-radioactive method.

Cytomegalovirus (CMV) is an important pathogen in persons who are immunocompromised or who have received organ transplants. The rate of symptomatic CMV infection is relatively higher after liver transplantation, and CMV hepatitis may be difficult to differentiate from rejection or cholangitis without recourse to liver biopsy and tissue culture. In this study we have used the polymerase chain reaction (PCR) to amplify and detect CMV-DNA sequences directly from the saliva and urine of liver transplant recipients. The procedure is simple, rapid and specific, and was found to be as sensitive as tissue culture for the diagnosis of CMV.

Base Sequence↗
PubMed1991

Direct evidence that ganglioside is an integral component of the thyrotropin receptor.

Gangliosides were extracted from purified human and porcine thyrotropin (TSH) receptors (TSH-R) and were detected by probing with an 125I-labeled sialic acid-specific lectin, Limax flavus agglutinin. Gangliosides copurified with human and porcine TSH-R migrated between monosialoganglioside GM1 and disialoganglioside GD1a. Ceramide glycanase digestion of the purified human TSH-R-associated glycolipid confirmed its ganglioside nature. It was resistant to Vibrio cholerae sialidase, which digests all gangliosides except GM1, but was sensitive to Arthrobacter ureafaciens sialidase, which digests all gangliosides including GM1. These findings indicate that the human TSH-R contains ganglioside that belongs to the galactosyl(beta 1----3)-N-acetylgalactosaminyl (beta 1----4)-[N-acetylneuraminyl(alpha 2----3)]galactosyl(beta 1----4) glucosyl(beta 1----1)ceramide (GM1) family. Its intimate association with receptor protein implies a key role for ganglioside in the structure and function of the TSH-R.

Adrenal Glands↗
PubMed1991

Overexpression of class I major histocompatibility complex accompanies insulitis in the non-obese diabetic mouse and is prevented by anti-interferon-gamma antibody.

Overexpression of class I major histocompatibility complex (MHC) proteins on pancreatic islet cells is a characteristic of autoimmune Type 1 (insulin-dependent) diabetes mellitus in humans and in animal models. Studies of post-mortem pancreases from humans with Type 1 diabetes suggest that overexpression of class I MHC proteins may precede mononuclear cell infiltration of the islets (insulitis). Pancreatic histology from the earliest stages of human Type 1 diabetes is rarely available. We have used the non-obese diabetic mouse, given cyclophosphamide to accelerate Beta-cell destruction, to investigate the temporal relationship between the overexpression of class I MHC protein and mRNA and other pathological changes associated with Beta-cell destruction. Prior to cyclophosphamide, immunoperoxidase staining showed that expression of class I MHC proteins was greater on islet cells and infiltrating inflammatory cells of the non-obese diabetic mouse than on islet cells of other mouse strains, whereas staining on exocrine cells was similar. On day three after cyclophosphamide administration, when insulitis had regressed, islet class I MHC protein expression had diminished. A dramatic increase in class I MHC protein expression occurred between days seven and nine, concomitant with reinfiltration of the islets by mononuclear cells; overexpression was seen both on islet cells and on surrounding exocrine cells, but only in the presence of mononuclear cell infiltration. By day 21, class I MHC protein overexpression was again confined to the islets, the exocrine pancreas being free of infiltration. Class I mRNA also increased dramatically by day eight but had virtually returned to normal by day 12.2+ effected by cytokines secreted by activated immuno-inflammatory cells. Class I MHC overexpression should enhance targeting of cytotoxic T cells to Beta cells bearing autoantigen.

Animals↗
PubMed1991

Factors predicting residual beta-cell function in the first year after diagnosis of childhood type 1 diabetes.

Twenty-five children aged 2-14 years (mean age 8.39 +/- 0.78 years) were studied prospectively during the first year after the diagnosis of type 1 diabetes. Of their clinical and metabolic features at diagnosis, only age showed a significant independent relationship with endogenous C-peptide production during the first year. Age was correlated with higher values for basal and stimulated plasma C-peptide at 7-14 days after diagnosis, at 6 months and at 12 months. At diagnosis, age was also associated with a higher value for HbA1c and a lower prevalence of insulin antibodies. C-peptide production peaked at 3 months and thereafter declined. Mean HbA1c and insulin requirement were both minimal at 6 months. At diagnosis, there were significant inverse relationships between basal C-peptide production and both insulin dose and HbA1c and between stimulated C-peptide production and HbA1c. Basal and stimulated C-peptide production were inversely related to insulin dose at 6 and 12 months. Stimulated C-peptide was higher at 12 months in children retaining islet cell antibodies. These findings confirm the importance of age as a predictor of residual beta-cell function in type 1 diabetes and indicate that older children present clinically following a slower course of beta cell destruction.

Adolescent↗
PubMed1991

Plasma cortisol and 11-deoxycortisol activity in depressed patients and normal volunteers.

Plasma cortisol and 11-deoxycortisol were measured in 30 depressed patients and 110 normal volunteers before and after a 1.0 mg dexamethasone suppression test (DST). Post-dexamethasone plasma cortisol, 11-deoxycortisol and the cortisol/11-deoxycortisol ratio were significantly higher in the depressives compared to the controls, even when age and sex were taken into account. Pre-dexamethasone plasma cortisol, post-dexamethasone cortisol, 11-deoxycortisol and their ratio were significantly higher in the cortisol nonsuppressors than in the suppressors. The measurement of post-dexamethasone 11-deoxycortisol and the ratio did not differentiate between endogenous and reactive depression. Using the normative data, we explored several methods for determining a criterion value to define abnormal post-dexamethasone plasma 11-deoxycortisol and the cortisol/11-deoxycortisol ratio in depressed patients. All showed poor sensitivity and a low positive predictive value for depression. The measurement of 11-deoxycortisol thus does not enhance the clinical utility of the DST.

Adult↗
PubMed1991

Reduction in insulitis following administration of IFN-gamma and TNF-alpha in the NOD mouse.

In insulin dependent diabetes mellitis (IDDM) beta cell destruction is associated with infiltration of the pancreatic islets by T lymphocytes and macrophages. Cytokine products from the infiltrating immunocytes not only have powerful immunoregulatory actions but also are capable of impairing islet cell functions and have thus been postulated to assume a central role in mediating anti-beta cell immunity and beta cell destruction. In an effort to explore further the role of cytokines in the pathogenesis of IDDM, we examined clinical, metabolic and pathological features of NOD/Wehi mice injected intraperitoneally with multiple doses of IFN-gamma and/or TNF-alpha. Blood glucose profiles were not significantly altered by injection of cytokines alone or in combination. Except for a hypoglycaemic rebound in mice injected with TNF-alpha, arginine stimulation tests revealed no disturbances in islet secretory function in cytokine injected mice. Compared with vehicle and cytokines alone, injection of IFN-gamma + TNF-alpha was associated with a variety of clinical and pathological changes including abdominal distention, piloerection, ascites, oedema, thymic atrophy, splenic enlargement and pancreatic distention. Histological examination of the pancreas in these mice revealed moderate to severe pancreatitis which included focal haemorrhagic necrosis, oedema and polymorphonuclear and mononuclear cell infiltration. The islets in these mice appeared normal morphologically and when stained for insulin. The injection of IFN-gamma + TNF-alpha, and to a lesser extent TNF-alpha alone, was associated with a significant reduction in the severity of insulitis. Examination of pancreatic MHC-class I and class II molecule expression revealed in mice given IFN-gamma + TNF-alpha, as compared with controls, significant and uniform induction of both these molecules on ductal and acinar cells; low level MHC-class II expression was also detectable on beta cells in these mice. MHC-class I molecules which were expressed at high levels by beta cells in control mice did not appear to change following administration of the cytokines alone or in combination. We conclude that despite their immunostimulatory actions in vitro and in other models in vivo, systemic administration of the cytokines IFN-gamma and/or TNF-alpha to NOD/Wehi mice does not activate or enhance, and may actually suppress, anti-beta cell immunity in this model.

Animals↗
PubMed1991

Characterization of pancreatic T lymphocytes associated with beta cell destruction in the non-obese diabetic (NOD) mouse.

Pancreatic beta cell destruction in the non-obese diabetic (NOD) mouse is mediated by T lymphocytes and macrophages and accelerated by cyclophosphamide. We purified pancreatic T lymphocytes from the NOD mouse for comparative phenotypic and functional analysis with T lymphocytes from spleen, peripheral blood and regional lymph nodes. Pancreatic T lymphocytes from NOD-Wehi mice, which have an incidence of spontaneous diabetes of less than 5%, had a CD4:CD8 ratio of 1.25 +/- 0.23 compared with 2.44 +/- 0.31 for peripheral blood lymphocytes. After cyclophosphamide, the CD4:CD8 ratio of pancreatic lymphocytes increased to 2.30 +/- 0.24 at day 7. T lymphocytes bearing IL-2 receptors increased two- to three-fold in number and their secretion of GM-CSF/IL-3 and IFN-gamma increased to a maximum on day 7. Pancreatic insulin content and mRNA levels declined sharply between days 10 and 12, at which time the majority of pancreatic T lymphocytes in hyperglycaemic mice were CD8+ (CD4:CD8 ratio 0.63 +/- 0.04 compared to 4.14 +/- 1.05 in peripheral blood). The pancreatic T lymphocyte CD4:CD8 ratio in prediabetic NOD-Lt mice, which have an incidence of spontaneous diabetes of about 60% at 150 days, was similar to that in untreated NOD-Wehi mice, but 25% of their pancreatic CD8 T lymphocytes were IL-2-receptor positive. Thus, significant changes in the phenotype of NOD pancreatic T lymphocytes following cyclophosphamide were not reflected in peripheral blood or spleen T lymphocytes. The earliest change after cyclophosphamide was an increase in activated, predominantly CD4+ T lymphocytes; with the development of beta cell destruction and hyperglycaemia, pancreatic T lymphocytes were, as in human IDDM, predominantly CD8+.

Animals↗
PubMed1991

Immunoblotting for the detection of TSH receptor autoantibodies.

Immunoblotting was optimized to detect autoantibodies to TSH receptors from human and porcine thyroid tissue and to determine their epitope specificity. Autoantibodies to putative TSH receptor proteins in thyroid particulate membranes were detected in approximately 35% of sera from patients with Graves' disease. However, despite modifications to increase immunoblotting sensitivity and specificity, only a minority (less than 15%) of Graves' disease sera contained autoantibodies that identified epitopes within TSH affinity-purified human or porcine receptor proteins. In these sera there was no correlation between the TSH receptor antibody titre, determined by radioreceptor assay, and receptor epitope reactivity. The sensitivity of immunoblotting was limited by reduced transfer of purified receptor from the gel. However, in addition, the inability to immunoblot the purified receptor with a majority of Graves' sera, under conditions designed to enhance receptor renaturation, appears to reflect a strict conformational requirement for immunoreactivity. Immunoblotting of purified receptors therefore has a limited application in detecting, and defining the epitope reactivity of, TSH receptor autoantibodies.

Animals↗
PubMed1991

Essential role for interferon-gamma and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice.

Experimental studies in vitro suggest that cytokines are important mediators in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). However, there is little evidence for the role of cytokines in vivo, either in humans or in the spontaneous animal models of IDDM such as the NOD mouse or BB rat. To address this question, we used the model of cyclophosphamide (CYP)-induced autoimmune diabetes in the NOD/Wehi mouse to examine for (a) the production of IFN-gamma and IL-6 from isolated islets, and (b) the effect of anti IFN-gamma or anti IL-6 monoclonal antibodies on the development of diabetes. After cyclophosphamide, the majority of these mice develop of mononuclear cell infiltrate (insulitis) which by 10-14 d is associated with beta cell destruction. IFN-gamma activity at low levels (2.7 +/- 0.3 U/ml) could be detected only in culture supernatants from islets isolated at day 7 post-cyclophosphamide. In contrast, IL-6 activity progressively increased from 457 +/- 44 U/ml at day 0 to 6,020 +/- 777 U/ml at day 10. Culture of islets with anti-CD3 monoclonal antibody resulted in a significant increase in IFN-gamma activity from 41 +/- 7 U/ml at day 0 to 812 +/- 156 U/ml at day 10. Mice given either anti-IFN-gamma or anti-IL-6 antibody had a significantly reduced (P less than 0.001) incidence of diabetes and especially with IFN-gamma, decreased severity of insulitis. We conclude that IFN-gamma and IL-6 have essential roles in the pathogenesis of pancreatic islet beta cell destruction in this model.

Animals↗
PubMed1991

Reactivity to human islets and fetal pig proislets by peripheral blood mononuclear cells from subjects with preclinical and clinical insulin-dependent diabetes.

A simple, direct assay for T-lymphocyte reactivity to islet antigen(s) in human insulin-dependent diabetes mellitus (IDDM) should facilitate preclinical diagnosis and the evaluation of intervention therapy to avert autoimmune-mediated beta-cell destruction. In subjects with preclinical or clinical IDDM, we measured the reactivity of peripheral blood mononuclear cells (PBMCs) incubated over 6 days with either adult human islets or fetal pig proislets, or other fetal pig tissues, and with human insulin. With islets, the stimulation index (SI) of [3H]thymidine uptake by PBMCs exceeded the mean + 2SD of control subjects in 6 of 6 preclinical subjects (SI 8.7 +/- 3.7), 7 of 11 clinical subjects (SI 5.2 +/- 3.4), and 1 of 12 control subjects (SI 2.7 +/- 1.7); with insulin, the responses were less in frequency and magnitude, being 4 of 6 (2.7 +/- 1.6), 3 of 11 (2.2 +/- 1.1), and 0 of 12 (1.20 +/- 0.55), respectively. The mean responses to islets of PBMCs from preclinical and clinical subjects differed significantly from control subjects (P less than 0.02 by 2-tailed Kruskal-Wallis test). Secretion of granulocyte macrophage colony-stimulating factor by PBMCs over 6 days was assayed in the preclinical group and generally paralleled the uptake of [3H]thymidine. PBMC reactivity to islets appeared to be at least as sensitive a marker of preclinical IDDM as autoantibodies to a 64,000-Mr protein, presumably the enzyme glutamic acid decarboxylase, in fetal pig proislets. In conclusion, islet-reactive T lymphocytes in subjects with preclinical and clinical IDDM can be identified in bulk culture of PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1990

Stabilization of glucose transporter mRNA by insulin/IGF-1 and glucose deprivation.

Chronic exposure of L6 myocytes to insulin/IGF-1 or glucose deprivation results in an increase in the level of brain-type glucose transporter (GLUT1) mRNA. We have investigated the effects of insulin and glucose deprivation on GLUT1 mRNA stability. The half-life of GLUT1 mRNA in control cells was 2-2.5 h. Insulin increased GLUT1 mRNA levels by 5- to 10-fold, and GLUT1 mRNA half-life and transcription by 2-fold. Glucose deprivation increased GLUT1 mRNA level by 2- to 4-fold and half-life by 2-fold. The effects of insulin and glucose deprivation on GLUT1 mRNA stability were additive. Cycloheximide partially blocked the induction of GLUT1 mRNA by insulin but not by glucose deprivation. GLUT1 mRNA was decreased to basal levels within 12h following insulin withdrawal or glucose refeeding. Cycloheximide did not block this de-induction, suggesting that insulin and glucose deprivation do not increase GLUT1 mRNA expression by inhibiting the synthesis of ribonucleases. These findings indicate that insulin/IGF-1 increases both GLUT1 mRNA stability and transcription by both protein synthesis-dependent and independent mechanisms, whereas glucose deprivation enhances GLUT1 mRNA stability by mechanisms independent of de novo protein synthesis.

Animals↗